Printable logic circuits comprising self-assembled protein complexes

This paper describes the fabrication of digital logic circuits comprising resistors and diodes made from protein complexes and wired together using printed liquid metal electrodes. These resistors and diodes exhibit temperature-independent charge-transport over a distance of approximately 10 nm and require no encapsulation or special handling. The function of the protein complexes is determined entirely by self-assembly. When induced to self-assembly into anisotropic monolayers, the collective action of the aligned dipole moments increases the electrical conductivity of the ensemble in one direction and decreases it in the other. When induced to self-assemble into isotropic monolayers, the dipole moments are randomized and the electrical conductivity is approximately equal in both directions. We demonstrate the robustness and utility of these all-protein logic circuits by constructing pulse modulators based on AND and OR logic gates that function nearly identically to simulated circuits. These results show that digital circuits with useful functionality can be derived from readily obtainable biomolecules using simple, straightforward fabrication techniques that exploit molecular self-assembly, realizing one of the primary goals of molecular electronics.


Materials and synthesis
The synthesis of PTEG-1 and PCBA are described elsewhere. 1,2 1-(3-(methoxycarbonyl)propyl) -1-phenyl [6.6 ]C 61 was obtained from Solenn B.V. Et 2 O as solvent was an analytical-grade reagent and was used as received. Other chemicals used in the synthesis were obtained from Sigma-Aldrich and TCI and used as received. The Ag TS substrates used in this work were made by mechanic template stripping as described else where; 3 we deposited 100 nm Ag (99.99 %) by thermal vacuum deposition onto a 3-inch wafer (without an adhesion layer). Using the UV-curable optical adhesive (OA) Norland 61, we glued 1 cm 2 glass chips on the metal surfaces. The Au mica substrates used in this work were made by thermally depositing 200 nm Au (99.99 %) in vacuum onto mica substrates at an annealing temperature of 350 • C. The substrates were gradually heated up to the annealing temperature over 1 h and kept at that temperature for 19 h until deposition; then the substrates were kept at the annealing temperature for another 2 h and allowed to gradually cooled down to room temperature. All substrates were used immediately after preparation.   The bright spots on the Au mica substrate correspond to the dust particles physisorbed onto the surface from the ambient environment during the fabrication of substrates, which were displaced during the fabrication of SAMs, as shown in Figure S1f and Figure S2. b, Plots of log | |versus potentials of Au mica (or Au Si )/PCBA//PSI//EGaIn junctions. c, Plots of log versus potential of Au mica (or Au Si )/PCBA//PSI//EGaIn junctions. Error bars represent 95% confidence intervals.

Simulation of logic circuits
The

Determination of thicknesses and PSI orientation
Our previous study on the self-assembled bilayers of fullerene derivatives with X-ray reflectivity confirmed that C 60 cages as anchoring groups contribute 0.9 nm to the thickness; 5 the distance from the bottom of C 60 fullerene to the carboxylic group of a PCBA molecule with minimized energy is 1.1 nm. The high yields of both EGaIn and CP-AFM junctions comprising SAMs of PCBA showed that the molecules are densely packed on the substrate. We therefore estimated the thickness of the SAMs of PCBA to be approximately 1 nm.
The structure of PSI trimers has been studied extensively by high-resolution X-ray techniques; 6-8 PSI trimer has a diameter of 21 nm and a maximal height of 9 nm. Because of its relatively large aspect ratio (i.e., the ratio between its height and diameter), height analysis by tapping mode AFM can provide evidence for the orientation of PSI trimers, e.g., side-oriented PSI trimers (the natural direction of electron flow inside the protein) is parallel to the substrate) is significantly taller than up-/down-oriented ones (the natural direction of electron flow is against/toward the substrate), and the formation of SAMs (e.g., aggregates of PSI trimers are beyond the dimensions of an individual protein). The combined results from height analysis and electrical characterization can effectively determine the exact orientation of PSI trimers, as demonstrated in our previous work.. 4,9 In this work, height analysis on the SAMs of PSI on PCBA with a Gaussian fit yields a statistical

Possible models for charge-transport mechanisms
Here, we considered charge-transport in the inverted Marcus region, via voltage-induced longrange coherent electron transfer, via flickering resonance, proton-coupled electron transfer for the charge-transport mechanism in PSI junctions.

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Charge-transfer in PSI that contributes to the charge recombination may operate in the inverted Marcus region to result in high internal quantum efficiency, i.e., charge recombination between the oxidized P700 cofactor and the reduced A1 cofactor is inhibited; 10 however, excitons were not present as the junctions comprising PSI SAMs in this work were characterized in the dark, and variable temperature measurements of the junctions showed that a thermally-activated charge-transfer necessary to generate a P700 + A1 − pair is absent. Therefore we do not believe that charge transfer in the inverted Marcus region is responsible for the asymmetric charge-transport across PSI junctions.
In the model of voltage-induced long-range coherent electron transfer developed by Naaman and coworkers, electronic states in the molecule are strongly coupled to their neighboring sites so that the density of states is nearly continuous to mediate temperature-independent resonant tunneling. 11  The flickering resonance model, developed by Beratan and coworkers, rationalizes that chargetransport over long distance in macromolecules can be mediated by the energy states generated from the fluctuations of the molecular structure and the medium, which come into resonance with the Fermi levels of the electrodes. 12 Although the model provides a good approximation on the exponential decay of current through macromolecules, the prerequisite fluctuations on the donor and acceptor unites in the molecules is temperature-dependent, which is not found in the variable temperature measurements of the PSI junctions. We therefore do not believe that the flickering resonance model applies to the charge-transport across PSI junctions.
Cahen and coworkers observed the transition from off-resonant to on-resonant tunneling across junctions comprising SAMs of metalloproteins, in which the spacer molecule modifies the S-24 coupling between the protein and the electrode to allows the states of cofactors containing Cu(II) ions to mediate efficient charge-transport. 13 In PSI trimers, the subunits containing metal ions (e.g., the P700 cofactor contains Mg cations and the F cofactor contains Fe cations) facilitate efficient charge-transfer via the electron transport chain by hopping; since the polarity of rectification of the PSI junctions opposes the redox gradient of the electron transport chain as shown in Supplementary Figure 9, the subunits do not seem to facilitate the asymmetric charge-transport across the PSI junctions. Both SAMs of PSI on PCBA and PCBM, despite the different orientation of PSI that may lead to varied protein-electrode coupling, facilitate efficient charge-transport with a tunneling decay coefficient smaller than 0.2 Å −1 , which suggests that long-range coherent tunneling is a property of folded polypeptides and not metalloproteins.
Proton-coupled electron transfer (PCET) describes the concerted (simultaneous) or sequential transfer of strongly-interacting protons and electrons from one atom to another, creating an energetically-favored pathway over decoupled electron and proton transfers in biological processes such as photosynthesis. 14,15 It can facilitate long-range and/or efficient charge transfer in proteins, e.g., proton-coupled electron transfers over 3-4 nm were observed on [FeFe]-hydrogenases, 16 and a near-zero tunneling decay coefficient for proton transfer was found on phenol-amines. 17 In contrast to solid-state large-area junctions comprising SAMs, molecular systems that exhibit PCET are usually characterized in solutions with varied pH. Although low activation energies were reported in several molecular systems that facilitate PCET, they unambiguously showed temperaturedependence on rate of proton/electron transfer which can be ascribed to i) the Boltzmann population of reactant proton vibrational states, ii) the thermal distribution of proton tunneling distances, and S-25 iii) the classical barriers for the vibronic transitions. 18,19 We cannot probe PCET through PSI SAMs using our experimental setups, but we discussed it here as a possible mechanism of charge-transport.
Whitesides and coworkers reported in a series of works that tunneling charge-transport is insensitive to the polarity of functional groups in thiol-SAMs, [20][21][22] in particular, the change in molecular dipole yields similar conductance in the SAMs and rectification ratios close to 1 at low biases (from −0.5 V to 0.5 V). These findings, together with the work by Baghbanzadeh and coworkers, 23,24 established that oligopeptides can mediate efficient tunneling charge-transport and the molecular dipole does not facilitate asymmetric charge-transport. These findings are not in conflict with our work. The dipoles that generate the electric field in PSI are not from individual peptides or polar groups, they are primarily the result of the alpha helices that surround the reaction center, which collectively exert a significantly larger influence than that of an oligopeptide or polar group. The reaction center itself is also polarized. Thus, it is the precise orientation of the dipoles within each PSI complex that gives rise to rectification, which is why we are able to observe it in single-complexes by CP-AFM, but to observe it in large-area junctions, most/all of the complexes must be oriented in the same direction so as not to cancel out. 4 We have previously observed vacuum level shifts induced by molecular dipoles in SAMs 25 that affect tunneling charge-transport 26 enough to induce rectification. 27 Thus, we are confident that sufficiently large (collective) dipole moments can affect charge-transport characteristics including rectification, and recognize the importance of contextualizing our observations with prior work to underscore that it is not as straightforward as simply installing dipole moments in a SAM and observing rectification. That distinction also supports our hypothesis that it is the loss of such alignment that eliminates rectification, while retaining S-26 efficient charge-transport, as shown by the control experiments (e.g., comparison between PCBA and PCBM linkers, and comparisons between PSI, BSA and denatured PSI).
There are examples of coherent tunneling transport further than 9 nm in molecular ensembles.
Slinker and coworkers reported effective coherent tunneling charge-transport across the SAMs of thiol-functionalized double-stranded DNAs with 100 and 17 base pairs on Au electrode, which resulted in a tunneling decay coefficient of 0.05 Å −1 over 34 nm. 28 In that work, the charge-transport properties of the SAMs were characterized by measuring the rate of charge transfer between the terminal Nile Blue redox probe and the Au electrode by cyclic voltammetry, in which the spatial separation of the probe and the electrode is defined by the length of DNA. Slinker and coworkers rationalized that the delocalized domains of the -stacked bases formed band-like structures that mediate efficient charge-transport. Saxena and coworkers reported coherent tunneling chargetransport across 5-20 nm in carbon-based large-area junctions comprising electrochemically grafted monolayers of nitroazobenznenes. 29 In this work, the lowest unoccupied molecular orbitals of the oligonitroazobenzenes were drawn to near-resonance by the electric field to facilitate activationless (or temperature-independent) coherent tunneling charge-transport at high bias.  Figure 25b and d), though we noticed that the skewness of the SAMs of PSI on PCBA and PCBM stabilized at 1.1 and 0 at higher biases (e.g., from −1.6 V to −2 V). The transition from negatively-skewed distribution to positively-skewed distribution observed in the SAMs S-28 of PSI on PCBA at low positive bias suggests the emergence of high-conductance tails with increasing bias and the alignment of molecular dipole and external field; the decrease in skewness in the SAMs of PSI on PCBM at high positive bias suggests the disappearance of high-conductance tail, and may correspond to the misalignment of molecular dipole and external field as the latter becomes dominant, in contrast to the more aligned SAMs of PSI on PCBA. The SAMs of PSI on PCBM showed a stronger bias-dependence of kurtosis than the SAMs of PSI on PCBA, with the convergence and divergence of conductance before and after 1.2 V. However, we cannot interpret the fluctuation in the skewness and kurtosis of the SAMs of PSI on PCBA and PCBM, since we cannot verify if it originated from the angular orientation of the dipole moment due to the complexity of PSI, particularly in bilayers with fullerene derivatives. Chen and coworkers applied this analysis to a much better-defined system in which the orientation of the dipoles with respect to the surface could be assumed with a reasonable degree of certainty. Regardless, the skewness and kurtosis of our ∕ data are consistent with a mechanism of charge-transport in which oriented dipoles partially mitigate conductance, further supporting the hypothesis that it is the (lack of) oriented dipoles that leads to (the lack of) rectification when PSI is (randomly) oriented by director In summary, we found the bias-dependence of skewness and kurtosis in the SAMs of PSI on PCBA, in which asymmetric charge-transport is mediated by the alignment of internal dipole and external field, and in the SAMs of FcC11SH and the SABs of PTEG-1, in which asymmetric charge-transport is dominated by tunneling-hopping mechanism. Though we observed unidirectional changes of these statistical moments at low bias, such patterns were lost for all investigated systems at an extended bias window. The work by Chen and coworkers showed the bias-dependence of skewness and kurtosis in symmetric charge-transport at low bias, but lacked insights into asymmetric charge-transport in large-area junctions over an extended range of bias. We therefore cannot correlate the skewness and kurtosis extrapolated from the molecular junctions reported in our work to the change of intermolecular dipole under external electric field.

Analysis on skewness and kurtosis
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